Full list of enriched GOs.

For understanding the evolutionary mechanism of sexually selected exaggerated traits, it is essential to uncover its molecular basis. By using broad-horned flour beetle that has male-specific exaggerated structures (mandibular horn, head horn and gena enlargement), we investigated the transcriptomic and functional characters of sex-biased genes. Comparative transcriptome of male vs. female prepupal heads elucidated 673 sex-biased genes. Counter-intuitively, majority of them were female-biased (584 genes), and GO enrichment analysis showed cell-adhesion molecules were frequently female-biased. This pattern motivated us to hypothesize that female-biased transcripts (i.e. the transcripts diminished in males) may play a role in outgrowth formation. Potentially, female-biased genes may act as suppressors of weapon structure. In order to test the functionality of female-biased genes, we performed RNAi-mediated functional screening for top 20 female-biased genes and 3 genes in the most enriched GO term (cell-cell adhesion, fat1/2/3, fat4 and dachsous). Knockdown of one transcription factor, zinc finger protein 608 (zfp608) resulted in the formation of male-like gena in females, supporting the outgrowth suppression function of this gene. Similarly, knockdown of fat4 induced rudimental, abnormal mandibular horn in female. fat1/2/3RNAi, fat4RNAi and dachsousRNAi males exhibited thick and/or short mandibular horns and legs. These cell adhesion molecules are known to regulate tissue growth direction and known to be involved in the weapon formation in Scarabaeoidea beetles. Functional evidence in phylogenetically distant broad-horned flour beetle suggest that cell adhesion genes are repeatedly deployed in the acquisition of outgrowth. In conclusion, this study clarified the overlooked functions of female-biased genes in weapon development.为解析性选择驱动的夸张性状的演化机制，阐明其分子基础至关重要。本研究以具有雄性特有的夸张结构（上颚角、头部角及颊部膨大）的宽角面粉甲虫为研究对象，对性别偏向基因（sex-biased genes）的转录组与功能特征展开了探究；通过对预蛹期头部组织开展雌雄比较转录组分析，共鉴定出673个性别偏向基因。出乎意料的是，其中绝大多数为雌性偏向基因（共584个），基因本体（Gene Ontology, GO）富集分析显示，细胞黏附分子（cell-adhesion molecules）常呈现雌性偏向表达。这一现象促使我们提出假说：雌性偏向转录本（即雄性中表达量下调的转录本）可能在突起形成过程中发挥作用，且雌性偏向基因或可作为雄性武器状结构的抑制因子。为验证雌性偏向基因的功能，我们针对排名前20的雌性偏向基因，以及富集程度最高的GO条目（细胞-细胞黏附，包含fat1/2/3、fat4及dachsous）中的3个基因，开展了RNA干扰（RNAi）介导的功能筛选；针对转录因子锌指蛋白608（zinc finger protein 608）的RNA干扰敲低实验，使雌性个体长出了雄性特有的颊部结构，证实了该基因的突起生长抑制功能。类似地，敲低fat4基因会导致雌性个体出现残留且形态异常的上颚角；经fat1/2/3、fat4及dachsous RNA干扰的雄性个体，其下颚角与足表现为粗壮且/或短小的表型。已知这类细胞黏附分子可调控组织生长方向，且在金龟子总科（Scarabaeoidea）甲虫的武器状结构形成过程中发挥作用；在系统发育亲缘关系较远的宽角面粉甲虫中获得的功能证据表明，细胞黏附基因在突起结构的演化获得过程中被反复招募利用。综上，本研究阐明了雌性偏向基因在雄性武器状结构发育中被忽视的功能。